α-Lipoic Acid Reduces Iron-induced Toxicity and Oxidative Stress in a Model of Iron Overload

Iron toxicity is associated with organ injury and has been reported in various clinical conditions, such as hemochromatosis, thalassemia major, and myelodysplastic syndromes. Therefore, iron chelation therapy represents a pivotal therapy for these patients during their lifetime. The aim of the present study was to assess the iron chelating properties of α-lipoic acid (ALA) and how such an effect impacts on iron overload mediated toxicity. Human mesenchymal stem cells (HS-5) and animals (zebrafish, n = 10 for each group) were treated for 24 h with ferric ammonium citrate (FAC, 120 µg/mL) in the presence or absence of ALA (20 µg/mL). Oxidative stress was evaluated by reduced glutathione content, reactive oxygen species formation, mitochondrial dysfunction, and gene expression of heme oxygenase-1b and mitochondrial superoxide dismutase; organ injury, iron accumulation, and autophagy were measured by microscopical, cytofluorimetric analyses, and inductively coupled plasma‒optical mission Spectrometer (ICP-OES). Our results showed that FAC results in a significant increase of tissue iron accumulation, oxidative stress, and autophagy and such detrimental effects were reversed by ALA treatment. In conclusion, ALA possesses excellent iron chelating properties that may be exploited in a clinical setting for organ preservation, as well as exhibiting a good safety profile and low cost for the national health system.


Introduction
Iron plays a pivotal role in various metabolic pathways encompassing a full range of cellular processes, such as energy metabolism and DNA synthesis, and serves as a cofactor for many enzymes, Plasma-Optical Emission Spectrometer (ICP-OES) assay ( Figure 1E). Consistent with these results, we also showed that iron overload resulted in a significant increase in ROS formation ( Figure 1F) when compared to untreated cells and such an increase was cancelled by concomitant treatment with ALA. Our results also showed that FAC treatment resulted in a significant mitochondrial impairment as measured by a significant reduction of Tu translation elongation factor, mitochondrial (TUFM) expression (Figure 2A-D). These results were further confirmed by cytofluorimetric analysis demonstrating a significant loss of mitochondrial membrane potential ( Figure 2E). Moreover, concomitant administration of ALA cancelled the detrimental effects of FAC when compared to FAC alone ( Figure 2). with ALA. Our results also showed that FAC treatment resulted in a significant mitochondrial impairment as measured by a significant reduction of Tu translation elongation factor, mitochondrial (TUFM) expression (Figure 2A-D). These results were further confirmed by cytofluorimetric analysis demonstrating a significant loss of mitochondrial membrane potential ( Figure 2E). Moreover, concomitant administration of ALA cancelled the detrimental effects of FAC when compared to FAC alone ( Figure 2).   . Perl's staining in untreated HS-5 cell line (A) and following treatment with FAC (120 µg/mL) alone (for 24 h) (B) and with ALA (20 µg/mL) alone (C) or in combination with FAC (D); intracellular iron concentration assessment (E); reactive oxygen species reduction following co-treatment of FAC plus ALA (*** p < 0.0001) at 2 h in HS-5 cell line vs. FAC alone (F). Results are expressed as median fluorescence intensity (** p < 0.001 vs. untreated control and *** p < 0.0001 vs. FAC alone). All values are presented as mean ± SE of four experiments in duplicate.
Increased oxidative stress following FAC treatment led to a significant increase in heme oxygenase 1 (HO-1) protein expression when compared to untreated cells and such an increase was prevented by concomitant treatment with ALA ( Figure 3A). These results were further confirmed by immunocytochemical analysis (Figure 3B-E). In addition, our results showed a significant increase in intracellular glutathione (GSH) content following FAC treatment when compared to untreated cells ( Figure 3F). Interestingly, co-treatment with ALA and FAC resulted in a further significant increase of GSH content when compared to FAC alone or untreated cells.
alone (for 24 h) (B) and with ALA (20 µg/mL) alone (C) or in combination with FAC (D); intracellular iron concentration assessment (E); reactive oxygen species reduction following co-treatment of FAC plus ALA (*** p < 0.0001) at 2 h in HS-5 cell line vs. FAC alone (F). Results are expressed as median fluorescence intensity (** p <0.001 vs. untreated control and *** p <0.0001 vs. FAC alone). All values are presented as mean ± SE of four experiments in duplicate.

Figure 2.
Immunofluorescences of TUFM localization in untreated HS-5 cell cultures (A) following FAC (120 µg/mL for 24 h) treatment alone (B) and with ALA (20 µg/mL) alone or in combination with FAC (C and D) and mitochondrial membrane depolarization evaluation (E). TUFM detection was Figure 2. Immunofluorescences of TUFM localization in untreated HS-5 cell cultures (A) following FAC (120 µg/mL for 24 h) treatment alone (B) and with ALA (20 µg/mL) alone or in combination with FAC (C,D) and mitochondrial membrane depolarization evaluation (E). TUFM detection was performed by incubation with anti-goat monoclonal antibody followed by secondary antibody conjugated to Rhodamine (red). Counterstaining of cells was performed by using the nuclear dye, DAPI (blue); (Scale bars 10 µm). Mitochondrial membrane depolarization evaluation after FAC treatment alone and in combination with ALA performed by FACS analysis (*** p < 0.0001 vs. FAC alone treatment). All values are presented as mean ± SE of four experiments in duplicate. performed by incubation with anti-goat monoclonal antibody followed by secondary antibody conjugated to Rhodamine (red). Counterstaining of cells was performed by using the nuclear dye, DAPI (blue); (Scale bars 10 µm). Mitochondrial membrane depolarization evaluation after FAC treatment alone and in combination with ALA performed by FACS analysis (*** p< 0.0001 vs. FAC alone treatment). All values are presented as mean ± SE of four experiments in duplicate.
Increased oxidative stress following FAC treatment led to a significant increase in heme oxygenase 1 (HO-1) protein expression when compared to untreated cells and such an increase was prevented by concomitant treatment with ALA ( Figure 3A). These results were further confirmed by immunocytochemical analysis (Figure 3B-E). In addition, our results showed a significant increase in intracellular glutathione (GSH) content following FAC treatment when compared to untreated cells ( Figure 3F). Interestingly, co-treatment with ALA and FAC resulted in a further significant increase of GSH content when compared to FAC alone or untreated cells.

In vitro Effect of α-Lipoic Acid on Iron Overload-mediated Autophagy
Consistent with previous reports, our results showed that iron overload following FAC treatment results in a significant increase of autophagy as measured by the AVO test when compared to untreated cells ( Figure 4A and B). Similar to oxidative stress results, co-treatment with FAC and ALA resulted in a significant reduction of autophagy when compared to FAC alone ( Figure 4A and B). These results were further confirmed by immunocytochemical analysis showing that FAC treatment resulted in a significant increase of Microtubule-associated protein 1A/1B-light chain 3

In Vitro Effect of α-Lipoic Acid on Iron Overload-mediated Autophagy
Consistent with previous reports, our results showed that iron overload following FAC treatment results in a significant increase of autophagy as measured by the AVO test when compared to untreated cells ( Figure 4A,B). Similar to oxidative stress results, co-treatment with FAC and ALA resulted in a significant reduction of autophagy when compared to FAC alone ( Figure 4A,B). These results were further confirmed by immunocytochemical analysis showing that FAC treatment resulted in a significant increase of Microtubule-associated protein 1A/1B-light chain 3 (LC3-II) when compared to controls ( Figure 4A-D) and such an effect was prevented when FAC and ALA were co-administered.

In vivo Effect of α-Lipoic Acid, Oxidative Stress, and Organ Injury
Consistent with the in vitro results, we also showed that FAC treatment in a zebrafish model resulted in a significant increase in liver and intestine iron storage ( Figure 5A and 5B) when compared to controls ( Figure 5). In addition, our data showed that concomitant treatment with ALA prevented an increase in iron content in all examined tissues ( Figure 5). Surprisingly, under our experimental conditions, no significant increase of iron storage was observed following FAC treatment ( Figure 5). Iron storage reduction following ALA treatment was even more evident compared to DFO treatment ( Figure 5A and 5B). Quantitative determination of iron (ICP-OES assay) showed that ALA reduced the iron storage in animals treated with ALA + FAC ( Figure 5C).
These results were further confirmed by ferroportin 1 (FPN1) expression showing that iron overload following FAC treatment resulted in a significant upregulation of gene expression in the

In Vivo Effect of α-Lipoic Acid, Oxidative Stress, and Organ Injury
Consistent with the in vitro results, we also showed that FAC treatment in a zebrafish model resulted in a significant increase in liver and intestine iron storage ( Figure 5A,B) when compared to controls ( Figure 5). In addition, our data showed that concomitant treatment with ALA prevented an increase in iron content in all examined tissues ( Figure 5). Surprisingly, under our experimental conditions, no significant increase of iron storage was observed following FAC treatment ( Figure 5). Iron storage reduction following ALA treatment was even more evident compared to DFO treatment ( Figure 5A,B). Quantitative determination of iron (ICP-OES assay) showed that ALA reduced the iron storage in animals treated with ALA + FAC ( Figure 5C).  These results were further confirmed by ferroportin 1 (FPN1) expression showing that iron overload following FAC treatment resulted in a significant upregulation of gene expression in the liver and intestine ( Figure 6C,F) when compared to controls. ALA treatment resulted in a significant decrease of FPN1 gene expression when compared to treatment with FAC alone. In addition, no significant changes were observed in FPN1 expression compared to both concentrations of DFO in the liver ( Figure 6F) whereas in the intestine, ALA resulted in a significant decrease of gene expression when compared to both DFO concentrations ( Figure 6A-C). Concomitantly to the above-presented results, we also showed that FAC treatment resulted in a significant increase of oxidative stress as measured by heme oxygenase 1b (HMOX1b, Danio rerio) and mitochondrial superoxide dismutase (mtSOD) gene upregulation in the intestine and liver ( Figure 6A,D). Our results again showed that ALA treatment resulted in a significant reduction of oxidative stress when compared to treatment with FAC alone. Finally, our results showed that oxidative stress reduction following ALA treatment was not significantly different when compared to DFO in the heart and liver, whereas it was significantly reduced in the intestine ( Figure 6). As a result of increased iron deposition and oxidative stress following FAC treatment, morphological analysis of the intestine demonstrated the presence of clear features of organ injury ( Figure 5). ALA treatment prevented organ injury in the intestine when compared to FAC treatment and histopathological recovery was significantly improved compared to DFO ( Figure 5). No significant morphological abnormalities were observed in the liver following all pharmacological treatments ( Figure 5).

Discussion
Iron overload may occur under various pathological conditions, including genetic forms, such as hereditary haemochromatosis, while others are acquired, such those related to repeated transfusions [17][18][19]. There are three chelating agents currently approved by the US Food and Drug Administration (FDA): Deferoxamine, deferiprone, and deferasirox. Iron chelators can reduce complications, such as cardiomyopathy, the major cause of death from iron overload. Furthermore, Figure 6. Effect of ALA on oxidative stress parameters of zebrafish liver and intestine. Gene expression analysis was performed following FAC treatment (120 µg/mL) alone and in combination with ALA (20 µg/mL) and DFO (131 µg/mL) for 48 h in zebrafish liver and intestine. HMOX1b, mtSOD (oxidative stress markers), and FPN1 levels were measured in the liver (A-C) and intestine (D-F) (HMOX1b: § § p < 0.001; mtSOD: ## p < 0.001; FPN1: *** p < 0.0001 vs. FAC treatment). Calculated value of 2 ∆∆Ct in untreated controls was 1. Data are expressed as mean ± SD of at least three independent experiments.

Discussion
Iron overload may occur under various pathological conditions, including genetic forms, such as hereditary haemochromatosis, while others are acquired, such those related to repeated transfusions [17][18][19]. There are three chelating agents currently approved by the US Food and Drug Administration (FDA): Deferoxamine, deferiprone, and deferasirox. Iron chelators can reduce complications, such as cardiomyopathy, the major cause of death from iron overload. Furthermore, iron chelation therapy can attenuate the progression of liver fibrosis and glucose intolerance in transfusion dependent patients [20,21]. However, in more than 10% of patients, the use of such agents is associated with adverse effects, such as retinal and auditory neurotoxicity, neutropenia and agranulocytosis, diarrhea, headache, nausea, abdominal pain, increased serum creatinine, and increased liver enzymes, rash, fatigue, and arthralgia [22]. Therefore, the aim of the present study was to test the effect of ALA in in vitro and in vivo models of iron overload with particular regard to its antioxidant and iron chelating properties.
Previous studies suggested that the accumulation of mitochondrial iron contributes to the decay of mitochondria and decreases life-sustaining functions, such as adenosine triphosphate (ATP) production, intracellular Ca 2+ buffering, regulation of cellular redox balance, and apoptosis [23,24]. Our in vitro results are consistent with these observations showing that FAC leads to intracellular iron accumulation, thus resulting in a significant impairment of mitochondrial membrane potential and organelle integrity, leading to ROS formation. Furthermore, our results showed that following administration of ALA, the levels of ROS gradually decreased toward basal conditions in mesenchymal stem cells, reducing intracellular iron content and restoring mitochondrial membrane potential and integrity. ALA is a dithiol compound normally bound to lysine residues of mitochondrial α-keto acid dehydrogenases; cytosolic and mitochondrial dehydrogenases rapidly reduce LA to dihydrolipoic acid (DHLA) [15]. Previous reports showed that ALA binds iron or any bivalent metal; hence, its property of iron chelation reduces the amount of free iron in the body, thereby alleviating oxidative stress, both enzymatically and by a free radical direct scavenging effect [15,25,26]. Interestingly, ALA is able to act inside the lysosomes, the conjugated action of cysteine and acid pH favors the rapid reduction of ALA in DHLA, possibly with the help of the lysosomal constituent, such as the lysosomal thiol reductase. Through its two vicinal thiolic groups, DHLA forms a  4 ), and a less stable one with Fe II [27][28][29][30][31][32][33]. Several groups have reported that lysosomal degradation of ferritin is crucial for the utilization of ferritin iron stores in a number of different settings and plays a central role in iron extraction from ferritin. In this process, it was shown that DHLA removes iron from ferritin in vitro [29]. Our results were further confirmed by HO-1 expression and intracellular GSH content. The GSH system is the most important cellular defense mechanism as a ROS scavenger regulating the intracellular redox state [34] and its synthesis is promptly activated by various oxidative triggers. In this regard, Macias-Barragan J et al. [35] showed that cell exposure to Cd 2+ resulted in a significant increase of GSH content as a result of the transcription activation of the enzymatic machinery for its biosynthesis. Furthermore, the authors showed that ALA also resulted in a significant increase of GSH content through the activation of the same pathway. Consistently with these observations, our results showed that iron overload following FAC treatment resulted in a significant increase in GSH content and that the concomitant treatment with ALA further increased such content when compared to FAC alone. Studies show ALA and DHLA may enhance cellular antioxidant defenses by a number of different mechanisms, including a direct antioxidant effect, and indirectly by augmenting the cellular GSH pool by increasing the expression of γ-glutamylcysteine ligase, the rate-controlling enzyme for GSH synthesis, Nrf2 activation [36]. In this regard, our resulted showed that HO-1, an NrF2 regulated protein involved in redox balance, is also upregulated following FAC treatment. Interestingly, co-treatment with FAC and ALA significantly reduced HO-1 expression when compared to FAC alone. These results may be dependent, at least in part, on the ability of ALA to increase intracellular GSH content, thus preventing NrF2 activation. Besides mitochondria, lysosomes are a major source of redox-active iron. Indeed, lysosomes are responsible for the autophagic degradation of iron-rich organelles, such as mitochondria and other metalloproteins. In this regard, our results also showed a significant increase in autophagy as measured by the increased number of autophagic granules following cytofluorimetric analysis and LC3 expression. Similar to our oxidative stress results, ALA treatment resulted in a significant decrease of autophagy. Finally, our in vitro 9 of 13 results were further confirmed in an in vivo model of iron overload. These results were consistent with the in vitro results, showing that ALA results in a significant reduction of iron storage in the liver and intestine and in the expression of FPN1. It is noteworthy that ALA resulted in a significant reduction of FPN1 expression when compared to a clinically relevant concentration of DFO. Furthermore, ALA, because of its direct and indirect antioxidant properties, resulted in a significant reduction of HMOX1b and mtSOD expression in the heart and intestine when compared to DFO.

PerlsSstaining
Perls staining (Bio-Optica, Milan, Italy) was performed according to the manufacturer's instructions. Briefly, tissue sections were passaged in distilled water and stained with Perls staining (Bio Optica, Milan, Italy) for 20 min. Sections were then rinsed in distilled water, dehydrated in ascending alcohols, cleared in xylene, and finally mounted for microscopic analysis.

Immunofluorescence
Cells were grown directly on coverslips before immunofluorescence. After washing with phosphate-buffered saline (PBS), cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, Milan, Italy) for 20 min at room temperature. After fixation, cells were washed three times in PBS for 5 min and blocked in Odyssey Blocking Buffer for 1 h at room temperature. Subsequently, the cells were incubated with primary antibody against HO-1 (anti-rabbit, Cat

Morphological Analysis
Zebrafish intestinal mucosa tissues were collected and fixed in 10% buffered-formaldehyde; after an overnight wash, specimens were dehydrated in graded ethanol and paraffin-embedded, preserving their anatomical orientation. Three to four micrometer thick sections were obtained according to routine procedures, mounted on sialane-coated slides and air-dried. Slides were dewaxed in xylene, hydrated using graded ethanol, and stained for histological studies (Hematoxylin and Eosin and Perls staining).

Iron Level Determination
The samples (cellular pellet or homogenate) were digested overnight with 200 µL of Nitric Acid 65%, Suprapur ® for trace analysis (Carlo Erba). After digestion, ultra-pure water (Merck) was added to the samples up to a volume of 2 mL and iron (Fe) was quantified with an Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES Optima 8000, Perkin Elmer, USA). Standards for the instrument calibration were prepared on the basis of mono-element certified reference solution ICP Standard (Merck) in the same acid matrix of the samples as well as the calibration blank. The method detection limits (MDL) estimated with 10 blanks was 5.4 µg/L, calculated according to the following equation: MDL= One-tailed student's t-test (p = 0.99%; df = n − 1) × Sr. A laboratory-fortified matrix (LFM) was determined as quality control and a recovery rate of 111% was obtained.

Statistical Analysis
Results are expressed as the means ± standard deviation (SD) of at least three independent experiments. Statistical analysis was carried out by one-way analysis of variance using the GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA, USA). Differences were considered significant at p < 0.05.

Conclusions
In conclusion, ALA may represent a valuable tool to be used in iron overload conditions because of its pleiotropic mechanisms of action, impacting on various, important pathophysiological mechanisms involved in cellular dysfunction and organ injury.